The first isolated human hepatoctyte 2D culture, in 1981, provided baseline protocols to support freshly isolated and immediately cultured populations. With cryopreservation technology, human hepatocytes are commercially-available on demand, however, about 75% of the world stores of cryopreserved human hepatocytes are below industry quality control standards and deemed non-useable. Creating methods to transition available but non-usable cryopreserved human hepatocytes into serviceable cell products is a primary goal. Using stable isotope (13C) resolved metabolomics (SIRM) applied to 2D cultures has revealed a major discovery that is contrary to metabolic paradigms: human hepatocytes do not metabolize the two primary carbon sources in basal media recipes, glucose and glutamine. When compared to rat hepatocytes in the starved state, it was concluded that human hepatocytes are in a stressed state that is probably catalyzed by surgery, death and the liver procurement process. The stressed hepatocytes consume internal proteins and lipid stores to generate glucose. In addition, nonhuman extracellular matrices are standard 2D culture conditions. Functionalizing these cells is based on two hypotheses. The first hypothesis: pyruvate and propionate are energy sources other than glucose and glutamine that are one enzyme step from the Krebs cycle, and will decrease lipolysis and proteolysis permitting amino acid and lipid stores to be diverted to proteins and membrane synthesis for increased cell attachment and viability. The second hypothesis is that natural biomatrices encountered by human hepatocytes will increase attachment once proteolysis is inhibited and protein synthesis of extracellular matrices is optimized. Two products are created from this study, (1) supporting media and extracellular matrices capable of transitioning non-usable human hepatocytes into useable cryopreserved hepatocyte products, and (2) a validated 13C SIRM method for contract research services using suspensions or attached human hepatocytes presently non-useable by conventional assays. The two specific aims will: (1) determine if addition of TCA nutrients (2-13C-pyruvate and 3-13C-propionate) increase human hepatocyte viability and/or attachment then (2) using the optimal media determined in specific aim 1, use the new basal media formulation to test three mixtures of human liver derived extracellular matrix for their efficacy at increasing viability and attachment. PUBLIC HEALTH RELEVANCE: In this grant application entitled "FUNCTIONALIZING NON-USEABLE CRYOPRESERVED HUMAN HEPATOCYTES", our studies catalog readily available, but currently ineffective, human hepatocytes as significant opportunities to transform subpar cellular products into serviceable in vitro human liver toxicodynamic and biokinetic models. This would be achieved by validating inactive and active metabolomic pathways using stable isotope (13C) resolved metabolomics (SIRM) that can demarcate needed cell eco-system supplements in support of culture success, and ultimately be applied in biokinetic models of human acute and chronic systems biology. This proposal will help to provide an abundance of cell material while decreasing developmental costs and time to commercialization.